Description: Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae). Amphidomataceae were found to be globally distributed in coastal waters and new areas of occurrence are regularly discovered. The AZA toxins accumulate mainly in shellfish and - when consumed by humans - can lead to the so-called azaspiracid shellfish poisoning syndrome (AZP). Given this serious threat to seafood production and to deepen knowledge about the distribution and risk potential of AZA-producing algae, an appropriate detection method enabling a fast identification and quantification for these toxigenic species is needed. Traditional light microscopy is time-consuming, requires expertise and is getting rather difficult when it comes to the detection, identification and quantification of small-sized plankton. To overcome this challenges, quantitative real-time PCR (qPCR) assays are increasingly used as a molecular additive. Basically, when amplifying the extracted DNA and using DNA standards, the amplification threshold (CT) gives information about the number of target species in the sample. For two AZA-producing species, Azadinium spinosum and Azadinium poporum, quantitative PCR assays have already been developed and successfully applied in the field. Another AZA-producing species, Amphidoma languida, was discovered in 2012 in Irish coastal waters and discovered as a new species within the group of Amphidomataceae - in close relationship with Azadinium spp. All available strains from Ireland, Iceland, Norway, Denmark and Spain produce azaspiracids. Moreover, Am. languida from the Atlantic coast of southern Spain was responsible for AZA amounts in shellfish above the EU regulatory limit, emphasizing the need for further investigations. We thus developed a quantitative TaqMan PCR assay, amplifying 60bp of the D2 region (located on the LSU/28S) of the ribosomal DNA (rDNA) to detect toxic Am. languida. To confirm assay specificity in vitro, cross-reactivity tests with DNA of a variety of related organisms were performed. This included 12 different Am. languida strains as positive controls, Amphidoma parvula, 10 Azadinium species (each including different strains), as well as 10 further related dinoflagellate species (Alexandrium spp., Gymnodinium spp., Heterocapsa spp., Karlodinium sp., Prorocentrum spp. & Scripsiella sp.). The developed probe and primer set successfully detected only A. languida strains. Currently, we perform tests of the newly-designed assay on spiked field samples to test and optimize the quantification ability of the assay. With this assay, we provide a tool for the rapid and distinctive quantification of the toxic dinoflagellate Amphidoma languida to be used in monitoring programs and bio-geographic studies.

Description: Amphidomataceae in the Labrador Sea and western Greenland waters Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents of shellfish poisoning. Over the last couple of years, four out of 27 described species of Amphidomataceae have been identified as primary source of AZA. Diversity and global biogeography of species of Amphidomataceae, however, still are poorly known. In summer 2017 we sampled the central Labrador Sea and the western Greenland coast from Gothaab Fjord (64° N) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed the presence of small Azadinium-like cells at fairly high densities of 9,200 cells L-1. Single cell isolation from that station yielded 14 clonal strains representing four different species, Azadinium obesum, Az. trinitatum, Az. dexteroporum, and a new species which is currently described taxonomically. For all cultured strains, no known AZA at measureable amounts were detected. From all stations along the cruise, filtered seawater samples were negative for AZA. Specific qPCR assays for DNA samples, targeting ribosomal genes of Azadinium and Amphidoma, revealed positive signals from the majority of stations along the Greenland west coast indicating a not yet recorded widespread occurrence, albeit at low densities, of Amphidomataceen species in that area. Solid phase adsorption toxin tracking (SPATT) samplers were long-term deployed during the expedition in a continuous water sampling system (FerryBox) and were negative for known AZA. The results highlight the presence of Amphidomataceae in the area but the lack of toxins in the field samples indicate a currently low risk of toxic Amphidomataceae blooms in arctic coastal waters.

Description: Planktonic dinophyte species of the familiy Amphidomataceae attract attention as producers of azaspiracids, lipophilic phycotoxins that accumulate in shellfish and cause human health problem for shellfish consumers. About 30 species are describe, which are small and difficult to differentiate with routine light microscopy. These group of species is thus an obvious case where molecular methods for detection and quantification are needed. An overview is given on the state of the art and current use of qPCR assays for Amphidomataceae (Dinophyceae). New challanges related to quantification and assay specificity due to the continuously increasing diversity of species and strains are highlighted and discussed.

Description: Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

Description: Species of the planktonic dinoflagellates Azadinium and Amphidoma are small, inconspicuous and difficult, if not impossible to be identified and differentiated by light microscopy. Within this group, there are some species that produce the marine biotoxin azaspiracid (AZA) while others are non-toxigenic, therefore a requirement exists for precise species identification. A quantitative polymerase chain reaction (qPCR) assay for molecular detection and quantification of one of the toxigenic species, Amphidoma languida, was designed and extensively tested. The assay was highly specific and sensitive to detect and quantify down to 10 target gene copies (corresponding to ca. 0.05 cells) per reaction. DNA cell quota and copy number cell−1 were constant for four different Am. languida strains, and for one strain they were shown to be stable at various time points throughout the growth cycle. Recovery of known cell numbers of Am. languida spiked into natural samples was 95–103%, and the assay was successfully tested on field samples collected from Irish coastal waters. This new qPCR assay is a valuable tool for routine monitoring for the prevention of AZA-shellfish-poisoning caused by the consumption of contaminated shellfish and is a supportive tool for studies on the biogeography of this AZA-producing species.

Description: Azaspiracids (AZA) are lipophilic marine biotoxins associated with shellfish poisoning which are produced by some species of Amphidomataceae. Diversity and global biogeography of this family are still poorly known. In summer 2017 plankton samples were collected from the central Labrador Sea and western Greenland coast from 64° N (Gothaab Fjord) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed small Azadinium-like cells (9200 cells l−1). Clonal strains established from plankton samples and scanning electron microscopy of fixed plankton samples revealed at least eight species of Amphidomataceae: Azadinium obesum, Az. trinitatum, Az. dexteroporum, Az. spinosum, Az. polongum, Amphidoma languida, Azadinium spec., and a new species described here as Azadinium perforatum sp. nov. The new species differed from other Azadinium species by the presence of thecal pores on the pore plate. All samples, including cultured strains, filtered seawater samples, and solid phase adsorption toxin tracking (SPATT) samplers deployed during the expedition in a continuous water-sampling system (FerryBox), were negative for AZA. DNA samples and PCR assays were positive for Amphidomataceae from most stations, whereas species-specific assays for three toxigenic species were rarely positive (two stations for Az. poporum, one station for Am. languida). The results highlight the presence of Amphidomataceae in the area but the lack of toxins and low abundance of toxigenic species currently indicate a low risk of toxic Amphidomataceae blooms in Arctic coastal waters.

Description: Shellfish contamination with azaspiracids (AZA), which are lipophilic marine biotoxins produced by marine dinoflagellates, is a major and recurrent problem for the Irish shellfish industry. AZA are produced by certain species of Amphidomataceae, but the species diversity of this group of microalgae in Irish waters is poorly known. Here we present a morphological and molecular characterization of multiple new strains of non-toxigenic Azadinium isolated on an oceanographic survey in 2018. A lack of AZA production for all strains presented here was demonstrated by LC-MS/ MS analysis. One strain of Azadinium caudatum var. margalefii (first strain for the area) confirmed nontoxigenicity of Atlantic populations of this species. One strain designated as Azadinium cf. zhuanum was similar to Az. zhuanum described from China but differed from the type strain in nucleus position, by the dominant number of apical plates, and by significant differences in rRNA gene sequences. Finally, two new non-toxigenic Azadinium species are described from the North East Atlantic: Azadinium galwayense sp. nov. and Azadinium perfusorium sp. nov. Azadinium galwayense differed from other Azadinium by a characteristic combination regarding presence and location of the ventral pore (vp; on the right side of the pore plate), of a pyrenoid (located in the episome), and by a pentagonal shape of the median anterior intercalary plate 2a, and lack of contact between plates 1´´ and 1a. Azadinium perfusorium shared the same vp position as Az. galwayense and differed by a characteristic combination of a pyrenoid located in the hyposome, a tetragonal shape of plate 2a, and a relatively large size of the two lateral anterior intercalary plates. Molecular phylogeny confirmed the distinctiveness of these two new species and their placement in Azadinium. The present findings significantly increased knowledge on the diversity of Azadinium species in the North East Atlantic.

Description: Numerous potentially toxic plankton species commonly occur in the Black Sea, and phycotoxins have been reported. However, the taxonomy, phycotoxin profiles, and distribution of harmful microalgae in the basin are still understudied. An integrated microscopic (light microscopy) and molecular (18S rRNA gene metabarcoding and qPCR) approach complemented with toxin analysis was applied at 41 stations in the northwestern part of the Black Sea for better taxonomic coverage and toxin profiling in natural populations. The combined dataset included 20 potentially toxic species, some of which (Dinophysis acuminata, Dinophysis acuta, Gonyaulax spinifera, and Karlodinium veneficum) were detected in over 95% of the stations. In parallel, pectenotoxins (PTX-2 as a major toxin) were registered in all samples, and yessotoxins were present at most of the sampling points. PTX-1 and PTX-13, as well as some YTX variants, were recorded for the first time in the basin. A positive correlation was found between the cell abundance of Dinophysis acuta and pectenotoxins, and between Lingulodinium polyedra and Protoceratium reticulatum and yessotoxins. Toxic microalgae and toxin variant abundance and spatial distribution was associated with environmental parameters. Despite the low levels of the identified phycotoxins and their low oral toxicity, chronic toxic exposure could represent an ecosystem and human health hazard.

Description: The Prorocentrales are a unique group of dinophytes based on several apomorphic traits, but species delimitation is challenging within the group. Prorocentrum triestinum was described by Josef Schiller in 1918 as an important bloomforming species from Trieste (Mediterranean, Adriatic Sea) with a conspicuous asymmetric outline and a small, asymmetrically located subapical spine. All subsequent records under this name fail to conform to Schiller’s original description. These inconsistencies have their origin in John Dodge’s 1975 revision of Prorocentrum, which placed Prorocentrum redfieldii, a more symmetrical, slender species with a long apical spine, into synonymy under P. triestinum. To clarify this confusion, we collected samples at the type locality of P. triestinum in Trieste and established a strain that is morphologically consistent with the protologue and suitable for use in epitypification. Morphology and rRNA sequence data of this strain were compared with four new strains identified as P. redfieldii from the Mediterranean Sea and the North Atlantic Ocean. Cells of P. triestinum had an asymmetric outline in lateral view and a small, dorso-subapical spine. These features, which are readily resolved by light microscopy, were distinct from those of the nearly symmetrical and slender cells of P. redfieldii, which had a long, apically located spine. The species are nevertheless closely related and share an identical architecture of the periflagellar area with a distinctive, largely reduced accessory pore together with a very small platelet 7. This apomorphy clearly differentiates both species from other species of Prorocentrum. Both species differ in their primary rRNA sequences, and ITS and LSU sequence differences will enable them to be distinguished in future meta-barcoding studies. The present study demonstrates that P. triestinum and P. redfieldii are distinct species and thus contributes to a reliable biodiversity assessment of Prorocentrum.